Fig. 5

Formation and growth of old-mouse organoids is reversed by inhibition of TGF-β. a Old-mouse organoids treated with or without 0.5 μM SB431542, a TGF-β inhibitor. Organoids were cultured at a density of 500 crypts per well in the organoid culture (passage 0). Data are reported as the mean ± standard deviation (SD) of 15 wells (5 wells per mouse; 3 mice per group). b Representative images of the indicated groups on day 5 of passage 0 after seeding (Scale bar = 200 μm). c Relative change in the organoid area on days 4 and 5 of passage 0. The percentages are relative to the old-mouse organoids on day 4. Data are shown as the mean ± SD of 15 organoids (5 organoid per mouse; 3 mice per group). Data are reported as the mean ± SD. d ID1, ID2, and ID3 mRNA expression in old-mouse organoids with and without 0.5 μM SB431542. Data are reported as the mean ± SEM (standard error of the mean) (n = 5). e Protein expression levels of ID1, ID2, ID3, p16^INK4a, and β-actin in old-mouse organoids with and without SB431542. The images were cropped from the original blot image in Supplementary Fig. 7a. The blot was cut before hybridization with antibodies. f Images of young-mouse organoids treated with or without various concentrations of TGFβ (0, 125, 250, 500, 1000 pg/mL) (Scale bar = 200 μm). The organoids were cultured at a density of 500 crypts per well in the organoid culture (passage 0) (n = 5 wells per mouse; n = 3 mice per group). g Number of organoid per crypt at passage 0 on day 4 ^*p < 0.05, ^**p < 0.01, ^***p < 0.001,.^****p < 0.0001 by the Mann–Whitney t-test. TGF-β1, transforming growth factor β1; ID1, inhitor of DNA binding 1; ID2, inhitor of DNA binding 2; ID3, inhitor of DNA binding 3